Enhanced Oral Bioavailability of β-Caryophyllene in Healthy Subjects Using the VESIsorb® Formulation Technology,a Novel Self-Emulsifying Drug Delivery System (SEDDS)

β-Caryophyllene (BCP), a common constituent of many spice and food plants, is gaining increased attention due to recent research identifying numerous potential health benefits. Due to limited oral bioavailability observed in preclinical models, the described benefits of BCP may be maximized by using a suitable delivery system. Additionally, human pharmacokinetics (PK) remain unknown. This study evaluates the relative oral bioavailability of BCP formulated in a self-emulsifying drug delivery system (SEDDS) based on VESIsorb^® formulation technology (BCP-SEDDS) compared to BCP neat oil. Hence, a randomized, double-blind, cross-over design, single oral dose study (100 mg BCP) in 24 healthy subjects (12 men/12 women) was performed under fasting conditions. Pharmacokinetic parameters were analyzed from individual concentration-time curves. The data show that BCP-SEDDS resulted in a 2.2/2.0-fold increase in AUC_0–12h/AUC_0–24h and a 3.6-fold increase in C_max compared to BCP neat oil. Moreover, BCP was absorbed faster from BCP-SEDDS (T_max: 1.43 h) compared to BCP neat oil (T_max: 3.07 h). Gender analysis revealed that there is no significant difference between men and women for both the investigated formulations and all investigated PK endpoints. In conclusion, BCP-SEDDS offers a well-tolerated and effective oral delivery system to significantly enhance the oral bioavailability of BCP in humans.     

Pharmacokinetic Characterization of LW6, a Novel Hypoxia-Inducible Factor-1α (HIF-1α) Inhibitor in Mice

LW6, an (aryloxyacetylamino)benzoic acid derivative, was recently identified to be an inhibitor of hypoxia-inducible factor-1α (HIF-1α), which is an attractive target for cancer therapeutics. Although LW6 is known to act by inhibiting the accumulation of HIF-1α, pharmacokinetics needs to be evaluated to assess its potential as an anti-tumor agent. Here, we investigated the plasma pharmacokinetics and metabolism of LW6 in mice. LW6 exhibited a small volume of distribution (0.5 ± 0.1 L/kg), and a short terminal half-life (0.6 ± 0.1 h). Following intravenous or oral administration, LW6 was rapidly converted to its active metabolite, (4-adamantan-1-yl-phenoxy)acetic acid (APA). Although LW6 was rapidly absorbed, its oral bioavailability, estimated using AUC_last values, was low (1.7 ± 1.8%). It was slowly degraded in mouse liver microsomes (t_1/2 > 1 h) and serum (t_1/2 > 6 h). About 54% or 44.8% of LW6 was available systemically as APA in the mouse after a single intravenous or oral administration, respectively. Thus, our results indicated the need to simultaneously consider the active metabolite as well as the parent compound for successful evaluation during lead optimization.     

A Newly Developed HPLC-UV/Vis Method Using Chemical Derivatization with 2-Naphthalenethiol for Quantitation of Sulforaphane in Rat Plasma

Sulforaphane (SFN), a naturally occurring isothiocyanate, has received significant attention because of its ability to modulate multiple biological functions, including anti-carcinogenic properties. However, currently available analytical methods based on high-performance liquid chromatography (HPLC)-UV/Vis for the quantification of SFN have a number of limitations, e.g., low UV absorbance, sensitivity, or accuracy, due to the lack of a chromophore for spectrometric detection. Therefore, we here employed the analytical derivatization procedure using 2-naphthalenethiol (2-NT) to improve the detectability of SFN, followed by HPLC separation and quantification with UV/Vis detection. The optimal derivatization conditions were carried out with 0.3 M of 2-NT in acetonitrile with phosphate buffer (pH 7.4) by incubation at 37 °C for 60 min. Separation was performed in reverse phase mode using a Kinetex C18 column (150 mm × 4.6 mm, 5 μm) at a flow rate of 1 mL/min, with 0.1% formic acid as a mobile phase A, and acetonitrile/0.1% formic acid solution as a mobile phase B with a gradient elution, with a detection wavelength of 234 nm. The method was validated over a linear range of 10–2000 ng/mL with a correlation of determination (R²) > 0.999 using weighted linear regression analysis. The intra- and inter-assay accuracy (% of nominal value) and precision (% of relative standard deviation) were within ±10 and <15%, respectively. Moreover, the specificity, recovery, matrix effect, process efficiency, and short-term and long-term stabilities of this method were within acceptable limits. Finally, we applied this method for studying in vivo pharmacokinetics (PK) following oral administration of SFN at doses of 10 or 20 mg/kg. The C_max (μg/mL), T_max (hour), and AUC_0–12h (μg·h/mL) of each oral dose were 0.92, 1.99, and 4.88 and 1.67, 1.00, and 9.85, respectively. Overall, the proposed analytical method proved to be reliable and applicable for quantification of SFN in biological samples.     

Comparative pharmacokinetics studies of benzoylhypaconine,benzoylmesaconine, benzoylaconine and hypaconitine in rats by LC‐MS method after administration of Radix Aconiti Lateralis Praeparata extract and Dahuang Fuzi Decoction

A rapid and sensitive high‐performance liquid chromatography–mass spectrometric (HPLC‐MS) method was developed and validated for simultaneous determination of benzoylhypaconine (BHA), benzoylmesaconine (BMA), benzoylaconine (BAC) and hypaconitine (HA) in rat plasma for the first time. The analytes were separated on a Kromasil C₁₈ column with a total running time of 11 min. The validation data demonstrated a sound feasibility for the newly developed method and it was then applied to the pharmacokinetic study of these analytes in rats. Pharmacokinetic behaviors of BHA, BMA, BAC and HA in rats were studied after oral administration of Radix Aconiti Lateralis Praeparata extract (FZ) and Dahuang Fuzi Decoction (DFD). The main parameters for the two groups of subjects were compared, and significant differences between Radix Aconiti Lateralis Praeparata extract group and Dahuang Fuzi Decoction group in calculated parameters, such as the area under the plasma concentration–time from zero to the last quantifiable time‐point (AUC_0–t), the area under the plasma concentration–time curve from zero to infinity (AUC_0–∞), peak plasma concentration (C_max), half‐life of elimination (T_1/2), mean retention time (MRT_0–t), plasma clearance (CL), volume of distribution (V_d) and time to reach C_max (T_max), were found. After oral administration of DFD, the AUC_0–t, AUC_0–∞ and C_max of BHA, BMA, BAC and HA decreased remarkably (p < 0.05) compared with those of the FZ extract group. V_d and CL values of BHA, BMA, BAC and HA increased, two of which showed significant difference (p < 0.05). T_1/2 and MRT_0–t values of BHA, BMA and BAC in the DFD group were significantly delayed compared with those of FZ extract group. Only the T_max of HA, the toxic ingredient in FZ, delayed significantly in DFD group compared with the value of FZ group. All these pharmacokinetic parameters were statistically compared, and the rationality of the combination for DFD was clearly demonstrated. Copyright © 2013 John Wiley & Sons, Ltd.     

Reductive Methylation of Homogeneous Primary β-Lauryl/myristyl 7/3 Polyethyleneoxy n = 3–18 Ethylamines under Phase-Transfer Catalysis Conditions

Homogeneous tertiary N,N-dimethyl-N-β-lauryl/myristyl 7/3 polyethyleneoxy n = 3–18 ethylamines, LM(EO)_nAT, are niche intermediates in the synthesis of homogeneous N-alkyl (C₁–C₁₈)-N,N-dimethyl-N-β-lauryl/myristyl 7/3 polyethyleneoxy n = 3–18 ethylammonium chlorides (unitary degree of oligomerization of ethylene oxide in the polyoxyethylene chain). This paper synthetically presents the dependence of the reductive methylation yields of homogeneous primary β-lauryl/myristyl 7/3 polyethyleneoxy n = 3–18 ethylamines, LM(EO)_nAP, on the reaction time (10–90 min), the temperature (70 °C), the molar ratio formic aldehyde /LM(EO)_nAP (1.1/1–2.5/1), the molar ratio HCOOH/LM(EO)_nAP (5/1), the degree of oligomerization of ethylene oxide in the homogeneous polyoxyethylene chain in the 3,6,9,12,18 series, and the structure of the phase-transfer catalysts. The steric effects of hydrophobic groups CH₃ and C₁₈H₃₇ grafted onto the ammonium function, and the micellar phenomena in the vicinity of their critical micellar concentration, directly proportional to the homogeneous degree of oligomerization, were highlighted. In all cases, a steady increase in reductive methylation yields was observed, with even quantitative values obtained. The high purity of the homologous series LM(EO)_nAT will allow their personalization as reference structures for the study of the evolution of basic colloidal characteristics useful in forecasting technological applications. LM(EO)_nAP were obtained either by direct amidoethylation (nucleophilic addition under basic catalysis of homogeneous lauryl/myristyl 7/3 polyethoxylated n = 3, 6, 9, 12, 18 alcohols, LM(EO)_nOH, to acrylamide monomer) or by cyanoethylation of LM(EO)_nOH under basic catalysis at 25–50 °C, in the presence of Fe²⁺ cations as oligomerization/polymerization inhibitor, followed by partial acid hydrolysis of homogeneous β-alkyl (C₁₂H₂₅/C₁₄H₂₉) 7/3 polyethyleneoxy n = 3, 6, 9, 12, 18 propionitriles, LM(EO)_nPN, to β-alkyl (C₁₂H₂₅/C₁₄H₂₉) 7/3 polyethyleneoxy n = 3, 6, 9, 12, 18 propionamides, LM(EO)_nPD, which led to LM(EO)_nAP by Hoffmann degradation. Homogeneous higher tertiary polyetheramines LM(EO)_nAT were structurally characterized.     

Randomized two‐way cross‐over bioequivalence study of two amoxicillin formulations and inter‐ethnicity pharmacokinetic variation in healthy Malay volunteers

The objectives of this study were to develop a new deproteinization method to extract amoxicillin from human plasma and evaluate the inter‐ethnic variation of amoxicillin pharmacokinetics in healthy Malay volunteers. A single‐dose, randomized, fasting, two‐period, two‐treatment, two‐sequence crossover, open‐label bioequivalence study was conducted in 18 healthy Malay adult male volunteers, with one week washout period. The drug concentration in the sample was analyzed using high‐performance liquid chromatography (UV–vis HPLC). The mean (standard deviation) pharmacokinetic parameter results of Moxilen® were: peak concentration (C_max), 6.72 (1.56) µg/mL; area under the concentration–time graph (AUC_0–8), 17.79 (4.29) µg/mL h; AUC_0–∞, 18.84 (4.62) µg/mL h. Those of YSP Amoxicillin® capsule were: C_max, 6.69 (1.44) µg/mL; AUC_0–8, 18.69 (3.78) µg/mL h; AUC0_0–∞, 19.95 (3.81) µg/mL h. The 90% confidence intervals for the logarithmic transformed C_max, AUC_0–8 and AUC_0–∞ of Moxilen® vs YSP Amoxicillin® capsule was between 0.80 and 1.25. Both C_max and AUC met the predetermined criteria for assuming bioequivalence. Both formulations were well tolerated. The results showed significant inter‐ethnicity variation in pharmacokinetics of amoxicillin. The C_max and AUC of amoxicillin in Malay population were slightly lower compared with other populations. Copyright © 2014 John Wiley & Sons, Ltd.     

MM quadruply bonded complexes supported by vinylbenzoate ligands: synthesis,characterization, photophysical properties and application as synthons

From the reactions between M₂(TⁱPB)₄ compounds and meta and para-vinylbenzoic acids (2 equiv.) in toluene at room temperature the compounds trans-M₂(TⁱPB)₂L₂, where L = m-vinylbenzoate 1A (M = Mo) and 1B (M = W) and TⁱPB = 2,4,6-triisopropylbenzoate, and where L = p-vinylbenzoate 2A (M = Mo) and 2B (M = W) have been isolated. Compounds 1A and 2A have been shown to undergo Heck carbon–carbon coupling reactions with phenyliodide to produce trans-Mo₂(TⁱPB)₂(O₂CC₆H₄-m-CHCH–C₆H₅)₂, 3A and trans-Mo₂(TⁱPB)₂(O₂CC₆H₄-p-CHCH–C₆H₅)₂, 4A. The molybdenum compounds 1A and 2A have been structurally characterized by single crystal X-ray crystallography. All the new compounds have been characterized by ¹H NMR, IR, UV-visible absorption and emission spectroscopy, high resolution MALDI-TOF MS, fs- and ns-transient absorption spectroscopy and fs-time-resolved IR spectroscopy. Electronic structure calculations employing density functional theory, DFT, and time-dependent DFT have been employed to aid in the interpretation of spectral data. All compounds show intense absorptions in the visible region corresponding to M₂δ to Lπ* charge transfer transitions. The lifetimes of the ¹MLCT state fall in the range of 1–10 ps and for the molybdenum complexes the T₁ states are ³δδ* with lifetimes ∼50 μs while for the tungsten complexes the T₁ are ³MLCT with lifetimes in the range of 3–10 ns.     

Cationic N,N-Dimethylglycine Ester Prodrug of 2R-α-Tocotrienol Promotes Intestinal Absorption via Efficient Self-Micellization with Intrinsic Bile Acid Anion

The intestinal absorption of hydrophobic compounds is severely influenced by their transportation rate through the unstirred water layer in the intestinal lumen. A member of the vitamin E family, α-Tocotrienol (α-T3) has remarkable pharmacological effects, but its intestinal absorption is hampered due to its hydrophobicity. Here, we prepared three ester derivatives of 2R-α-T3, and we selected a suitable prodrug compound using rat plasma and liver microsomes. The micellization profile of the selected compound in the presence of taurocholic acid (TCA) was evaluated. After gastrostomy administration of the prodrug candidate or α-T3 solution containing TCA, AUC values were determined for α-T3 in plasma obtained from bile duct-ligated rats. Among the three types in the efficiency of the reconversion to the parent drug, α-T3 N,N-dimethylglycinate (α-T3DMG) was the best prodrug; α-T3DMG formed mixed micelles via ion pairs with anionic TCA. The solubility of α-T3DMG in n-octanol/water depended on its ratio to TCA. The AUC after α-T3DMG administration to ligated rats was 2-fold higher than that after α-T3 administration, suggesting a smooth interaction with intrinsic bile acids. In conclusion, utilization of the prodrug synthesized using N,N-dimethylglycine ester may be a beneficial approach to promote intestinal absorption of α-T3 via self-micellization with intrinsic bile acid.     

Validation of an analytical LC-MS/MS method in human plasma for the pharmacokinetic study of atomoxetine

This study aimed to validate a sensitive and reliable analytical method for the pharmacokinetic study of atomoxetine in human plasma by liquid chromatography-electrospray ionization-tandem mass spectrometry. Metoprolol was used as an internal standard. After liquid-liquid extraction with methyl t-butyl ether, the supernatant was evaporated. The residue was then reconstituted and an aliquot was injected into the high performance liquid chromatographic system. Separation was performed on a Phenomenex Luna C₁₈ column (2.0 mm × 100 mm, 3 μm particles) with a mobile phase of 10 mM ammonium formate buffer: methanol = 10: 90 (v/v). Tandem mass spectrometry was performed in the electrospray ionization positive ion mode using the multiple reaction monitoring mode for quantification. The mass transition pairs of m/z 256 → 44 for atomoxetine and m/z 268 → 116 for the internal standard were used. The flow rate of the mobile phase was 0.25 mL/min and the retention times of atomoxetine and the internal standard were found to be 1.0 and 0.9 min, respectively. The calibration curve for atomoxetine was linear in the concentration range of 1–750 ng/mL (r ² = 0.9992) with a lower limit of quantification of 1 ng/mL. The mean accuracy for atomoxetine was 93–102%. The coefficients of variation (precision) in the intra- and inter-day validation for atomoxetine were 4.0–6.8 and 1.1–9.6%, respectively. The pharmacokinetic parameters of atomoxetine were evaluated after administration of a 40-mg single oral dose to twelve healthy male volunteers. The mean AUC_{0–24 h}, C _max, T _max and T _1/2 for atomoxetine were 1.9 ± 0.8 μg h/mL, 0.34 ± 0.11 μg/mL, 1.0 ± 0.5 h and 3.9 ± 1.3 h, respectively.     

Migratory insertion of isocyanide into a ketenyl–tungsten bond as key step in cyclization reactions

Treatment of the side-on tungsten alkyne complex of ethinylethyl ether [Tp*W(CO)₂(η²-C,C′-HCCOCH₂CH₃)]⁺ {Tp* = hydridotris(3,4,5-trimethylpyrazolyl)borate} (2a) with n-Bu₄NI afforded the end-on ketenyl complex [Tp*W(CO)₂(κ¹-HCCO)] (4a). This formal 16 ve complex bearing the prototype of a ketenyl ligand is surprisingly stable and converts only under activation by UV light or heat to form a dinuclear complex [Tp*₂W₂(CO)₄(μ-CCH₂)] (6). The ketenyl ligand in complex 4a underwent a metal template controlled cyclization reaction upon addition of isocyanides. The oxametallacycles [Tp*W(CO)₂{κ²-C,O-C(NHXy)C(H)C(Nu)O}] {Nu = OMe (7), OEt (8), N(i-Pr)₂ (9), OH (10), O_1/2 (11)} were formed by coordination of Xy-NC (Xy = 2,6-dimethylphenyl) at 4a and subsequent migratory insertion (MI) into the W-ketenyl bond. The resulting intermediate is susceptible to addition reactions with protic nucleophiles. Compounds 2a-PF₆, 4a/b, and 7–11 were fully characterized including XRD analysis. The cyclization mechanism has been confirmed both experimentally and by DFT calculations. In cyclic voltammetry, complexes 7–9 are characterized by a reversible W(ii)/W(iii) redox process. The dinuclear complex 11 however shows two separated redox events. Based on cyclic voltammetry measurements with different conducting electrolytes and IR spectroelectrochemical (SEC) measurements the W(ii)/W(iii) mixed valent complex 11⁺ is assigned to class II in terms of the Robin-Day classification.

The prototype ketenyl ligand is bound end-on despite a formal 16 valence electron count at the metal. This situation opens a reaction pathway for a multicomponent cyclization centred on the migration of the ketenyl ligand.     

3,4-Methylenedioxymethamphetamine (MDMA) and metabolites disposition in blood and plasma following controlled oral administration

3,4-Methylenedioxymethamphetamine (MDMA) is an illicit phenethylamine ingested for entactogenic and euphoric effects. Although blood is more commonly submitted for forensic analysis, previous human MDMA pharmacokinetics research focused on plasma data; no direct blood–plasma comparisons were drawn. Blood and plasma specimens from 50 healthy adult volunteers (33 males, 17 females, 36 African-American) who ingested recreational 1.0 and 1.6 mg/kg MDMA doses were quantified for MDMA and metabolites 4-hydroxy-3-methoxymethamphetamine (HMMA), 3,4-methylenedioxyamphetamine (MDA), and 4-hydroxy-3-methoxyamphetamine (HMA) by two-dimensional gas chromatography–mass spectrometry. Specimens were collected up to 3 h post-dose and evaluated for maximum concentration (C _max), first detection time (t _first), time of C _max (t _max), and 3-h area under the curve (AUC_0–3 h); as well as blood metabolite ratios and blood/plasma ratios. Median blood MDMA and MDA C _max were significantly greater (p?<?0.0005) than in plasma, but HMMA was significantly less (p?<?0.0005). HMA was detected in few blood specimens, at low concentrations. Nonlinear pharmacokinetics were not observed for MDMA or MDA in this absorptive phase, but HMMA C _max and AUC_0–3 h were similar for both doses despite the 1.6-fold dose difference. Blood MDA/MDMA and MDA/HMMA significantly increased (p?<?0.0001) over the 3-h time course, and HMMA/MDMA significantly decreased (p?<?0.0001). Blood MDMA C _max was significantly greater in females (p?=?0.010) after the low dose only. Low-dose HMMA AUC_0–3 h was significantly decreased in females’ blood and plasma (p?=?0.027) and in African-Americans’ plasma (p?=?0.035). These data provide valuable insight into MDMA blood–plasma relationships for forensic interpretation and evidence of sex- and race-based differential metabolism and risk profiles.

Metabolic Behaviors of Aconitum Alkaloids in Different Concentrations of Aconiti Lateralis Radix Praeparata and Effects of Aconitine in Healthy Human and Long QT Syndrome Cardiomyocytes

Aconiti Lateralis Radix Praeparata (Fu Zi) is the processed lateral root of Aconitum carmichaelii Debx, which is widely used in emergency clinics. Poisoning incidents and adverse reactions occur with the improper intake of Fu Zi. Metabolic characteristics of aconitum alkaloids of Fu Zi may vary, and the effects of Fu Zi in healthy and Long QT syndrome (LQTS) patients is unknown. In this experiment, 24 Sprague Dawley rats were randomly divided into three groups: 2.0, 1.0, and 0.5 g/kg dose groups, and blood samples were collected after the oral administration of Fu Zi extract. We used an ultra-high performance liquid chromatography-tandem mass spectrometry system to detect the concentrations of six aconitum alkaloids. Cell toxicity, calcium imaging, and patch-clamp recordings of human induced pluripotent stem cells-cardiomyocytes (hiPSC-CMs) of aconitine in healthy and LQTS were observed. We found that the AUC_(0–48h), C_max, and t_1/2 of the six compounds increased with the multiplicative dosages; those in the high group were significantly higher than those in the low group. Aconitine concentration-dependently decreased the amplitude, which has no significant effect on the cell index of normal hiPSC-CMs. Aconitine at 5.0 μM decreased the cell index between 5–30 min for LQTS hiPSC-CMs. Meanwhile, aconitine significantly increased the frequency of calcium transients in LQTS at 5 μM. Aconitine significantly shortened the action potential duration of human cardiomyocytes in both normal and LQTS groups. These results show metabolic behaviors of aconitum alkaloids in different concentrations of Fu Zi and effects of aconitine in healthy and LQTS patients.     

LC–MS Determination and Pharmacokinetic Study of Salidroside in Rat Plasma after Oral Administration of Traditional Chinese Medicinal Preparation Rhodiola crenulata Extract

A simple, rapid and sensitive liquid chromatography–mass spectrometry (LC–MS) method was developed for the quantification of salidroside in rat plasma and the study of its pharmacokinetics after oral administration of 15 g kg^?1 Rhodiola crenulata extract to Wistar rats. A 200 μL plasma sample was extracted by acetonitrile and performed on Kromasil C₁₈ column (150 mm × 4.6 mm, 5 μm) with the mobile phase of acetonitrile–water (11:89) within a run time of 8 min. The analyte was monitored with electrospray ionization (ESI) by selected ion monitoring (SIM) mode. The target ions were m/z 299.20 for salidroside and m/z 150.00 for internal standard (IS) paracetamol. A good linear relationship was obtained over the range of 100–20,000 ng mL^?1 and the lower limit of quantification was 100 ng mL^?1. The validated method was successfully applied for the pharmacokinetic study of salidroside in rat. After oral administration of Rhodiola crenulata extract, the main pharmacokinetic parameters T _max, T _1/2, C _max, AUC _0?t and AUC _0?∞ were 0.56 ± 0.21 h, 7.91 ± 4.42 h, 3,386 ± 2,138 ng mL^?1, 16,146 ± 6,558 ng h mL^?1 and 18,599 ± 6,529 ng h mL^?1, respectively.     

Biocatalytic cascades and intercommunicated biocatalytic cascades in microcapsule systems

Biomolecule-loaded nucleic acid-functionalized carboxymethyl cellulose hydrogel-stabilized microcapsules (diameter ca. 2 μm) are introduced as cell-like containments. The microcapsules are loaded with two DNA tetrahedra, T₁ and T₂, functionalized with guanosine-rich G-quadruplex subunits, and/or with native enzymes (glucose oxidase, GOx, and/or β-galactosidase, β-gal). In the presence of K⁺-ions and hemin, the T₁/T₂ tetrahedra constituents, loaded in the microcapsules, assemble into a hemin/G-quadruplex bridged tetrahedra dimer DNAzyme catalyzing the oxidation of Amplex Red to Resorufin by generating H₂O₂. In the presence of co-loaded GOx or GOx/β-gal, the GOx//T₁/T₂ hemin/G-quadruplex cascade catalyzing the glucose-mediated oxidation of Amplex Red to Resorufin, and the three-biocatalysts cascade consisting of β-gal//GOx//hemin/G-quadruplex bridged T₁/T₂ catalyzing the lactose-driven oxidation of Amplex Red to Resorufin proceed in the microcapsules. Enhanced biocatalytic transformations in the microcapsules, as compared to the performance of the reactions in a homogeneous phase, are observed, due to the proximity of the biocatalysts in a confined volume. As the synthetic methodology to prepare the microcapsules yields boundaries functionalized with complementary nucleic acid tethers, the dynamic association of different microcapsules, loaded selectively with biomolecular catalysts, proceeds. The dynamic dimerization of GOx-loaded microcapsules and hemin/G-quadruplex bridged T₁/T₂ DNAzyme-loaded microcapsules yields effective intercommunicated microcapsules driving the GOx//hemin/G-quadruplex bridged T₁/T₂ DNAzyme cascade. In addition, the dynamic dimerization of GOx-loaded microcapsules with β-gal//hemin/G-quadruplex bridged T₁/T₂-loaded microcapsules enables the bi-directional intercommunicated operation of the lactose-stimulated three catalysts β-gal//GOx//hemin/G-quadruplex bridged T₁/T₂ DNAzyme cascade. The guided separation and formation of dynamic supramolecular dimer microcapsular containments, and the dictated switchable operation of intercommunicated biocatalytic cascades are demonstrated.

Dynamic dimerization of GOx-loaded microcapsules with β-gal//hemin/G-quadruplex-bridged T₁/T₂-loaded microcapsules guides the bi-directional intercommunication of the three catalysts cascade.     

A Proteinaceous Alpha-Amylase Inhibitor from Moringa Oleifera Leaf Extract: Purification,Characterization, and Insecticide Effects against C. maculates Insect Larvae

The main objective of the current study was the extraction, purification, and enzymatic characterization of a potent proteinaceous amylase inhibitor from Moringa oleifera. The antimicrobial potential and insecticide effects against C. maculates insect larvae were also studied. The α-amylase inhibitor was extracted in methanol (with an inhibitory activity of 65.6% ± 4.93). Afterwards, the inhibitor αAI.Mol was purified after a heat treatment at 70 °C for 15 min followed by one chromatographic step of Sephadex G-50. An apparent molecular weight of 25 kDa was analyzed, and the N-terminal sequence showed the highest identity level (89%) with the monomeric α-amylase inhibitor from Triticum dicoccoides. αAI.Mol was found to tolerate pH values ranging from 5.0 to 11.0 and showed maximal activity at pH 9.0. Thermal stability was remarkably important, since the inhibitory activity was maintained at 55% after 1 h of incubation at 70 °C and at 53% after an incubation of 45 min at 80 °C. The potency of the current purified inhibitor against amylases from different origins indicates that αAI.Mol seems to possess the highest affinity toward human salivary α-amylase (90% inhibitory activity), followed by the α-amylase of insects Callosobruchus maculatus and Tribolium confusum (71% and 61%, respectively). The kinetic parameters were also calculated, and the K_max and V_max of the digestive amylase were estimated at 185 (mmol/min/mg) and 0.13 mM, respectively. The inhibitor possesses a strong bactericidal effect against Gram+ and Gram- strains, and the MIC values were >1 against B. cereus but >6 against E. coli. Interestingly, the rates of survival and pupation of C. maculates insect larvae were remarkably affected by the purified αAI.Mol from Moringa oleifera.     

Quantitative analysis and pharmacokinetic comparison of multiple bioactive components in rat plasma after oral administration of Qi-Shen-Ke-Li formula and its single-herb extracts using ultra-high-performance liquid chromatography–tandem mass spectrometry

Qi-Shen-Ke-Li (QSKL), a traditional Chinese formula prepared from six herbs, has long been used for the treatment of coronary heart disease and chronic heart failure. However, the herbal combination mechanism and underlying material basis of this multi-herbal formula are not clear. In this study, an ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method to simultaneously determine multiple bioactive compounds in QSKL was established and validated. Using the developed method, 18 bioactive components in rat plasma after oral administration of QSKL formula and its single herb extracts were quantified. Based on these results, pharmacokinetic (PK) parameters (T_1/2, T_max, C_max, AUC_0–48h, and AUC_0–∞) of the 18 bioactive components were analyzed and compared using PKSlover 2.0 PK software. The experimental data suggested that significant changes in PK profiles were observed between the QSKL formula and its single-herb extracts. The herbal combination in QSKL significantly influences the system exposure and the PK behaviors of the 18 bioactive components, indicating multicomponent interactions among the herbs. This study provides insight into the herbal combination mechanism and underlying material basis of the QSKL formula.     

Intermolecular CDC amination of remote and proximal unactivated Csp3–H bonds through intrinsic substrate reactivity – expanding towards a traceless directing group

An intermolecular radical based distal selectivity in appended alkyl chains has been developed. The selectivity is maximum when the distal carbon is γ to the appended group and decreases by moving from γ → δ → ε positions. In –COO– linked alkyl chains, the same distal γ-selectivity is observed irrespective of its origin, either from the alkyl carboxy acid or alkyl alcohol. The appended groups include esters, N–H protected amines, phthaloyl, sulfone, sulfinimide, nitrile, phosphite, phosphate and borate esters. In borate esters, boron serves as a traceless directing group, which is hitherto unprecedented for any remote C_sp³–H functionalization. The selectivity order follows the trend: 3° benzylic > 2° benzylic > 3° tertiary > α to keto > distal methylene (γ > δ > ε). Computations predicted the radical stability (thermodynamic factors) and the kinetic barriers as the factors responsible for such trends. Remarkably, this strategy eludes any designer catalysts, and the selectivity is due to the intrinsic substrate reactivity.

An intermolecular amination at the distal methylene carbon has been realized in an appended alkyl chain with electron withdrawing groups. Traceless remote C_sp³–H functionalization has been accomplished using borate esters.     

Synthesis and Antibacterial Properties of Oligomeric Dehydrogenation Polymer from Lignin Precursors

The lignin precursors of coniferin and syringin were synthesised, and guaiacyl-type and guaiacyl-syringyl-type oligomeric lignin dehydrogenation polymers (DHP and DHP-GS) were prepared with the bulk method. The carbon-13 nuclear magnetic resonance spectroscopy showed that both DHP-G and DHP-GS contained β-O-4, β-5, β-β, β-1, and 5-5 substructures. Extraction with petroleum ether, ether, ethanol, and acetone resulted in four fractions for each of DHP-G (C₁₁–C₁₄) and DHP-GS (C₂₁–C₂₄). The antibacterial experiments showed that the fractions with lower molecular weight had relatively strong antibacterial activity. The ether-soluble fractions (C₁₂ of DHP-G and C₂₂ of DHP-GS) had strong antibacterial activities against E. coli and S. aureus. The C₁₂ and C₂₂ fractions were further separated by preparative chromatography, and 10 bioactive compounds (G₁–G₅ and GS₁–GS₅) were obtained. The overall antibacterial activities of these 10 compounds was stronger against E. coli than S. aureus. Compounds G₁, G₂, G₃, and GS₁, which had the most significant antibacterial activities, contained β-5 substructures. Of these, G₁ had the best antibacterial activity. Its inhibition zone diameter was 19.81 ± 0.82 mm, and the minimum inhibition concentration was 56.3 ± 6.20 μg/mL. Atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) showed that the antibacterial activity of G₁ was attributable to a phenylcoumarin dimer, while the introduction of syringyl units reduced antibacterial activity.     

Cloning,Expression, Purification,and Characterization of β-Galactosidase from Bifidobacterium longum and Bifidobacterium pseudocatenulatum

Expression and purification of β-galactosidases derived from Bifidobacterium provide a new resource for efficient lactose hydrolysis and lactose intolerance alleviation. Here, we cloned and expressed two β-galactosidases derived from Bifidobacterium. The optimal pH for BLGLB1 was 5.5, and the optimal temperature was 45 °C, at which the enzyme activity of BLGLB1 was higher than that of commercial enzyme E (300 ± 3.6 U/mg) under its optimal conditions, reaching 2200 ± 15 U/mg. The optimal pH and temperature for BPGLB1 were 6.0 and 45 °C, respectively, and the enzyme activity (0.58 ± 0.03 U/mg) under optimum conditions was significantly lower than that of BLGLB1. The structures of the two β-galactosidase were similar, with all known key sites conserved. When o-nitrophenyl-β-D-galactoside (oNPG) was used as an enzyme reaction substrate, the maximum reaction velocity (V_max) for BLGLB1 and BPGLB1 was 3700 ± 100 U/mg and 1.1 ± 0.1 U/mg, respectively. The kinetic constant (K_m) of BLGLB1 and BPGLB1 was 1.9 ± 0.1 and 1.3 ± 0.3 mmol/L, respectively. The respective catalytic constant (k_cat) of BLGLB1 and BPGLB1 was 1700 ± 40 s⁻¹ and 0.5 ± 0.02 s⁻¹, respectively; the respective k_cat/K_m value of BLGLB1 and BPGLB1 was 870 L/(mmol∙s) and 0.36 L/(mmol∙s), respectively. The K_m, k_cat and V_max values of BLGLB1 were superior to those of earlier reported β-galactosidase derived from Bifidobacterium. Overall, BLGLB1 has potential application in the food industry.